This application relates to a method and apparatus for detecting optically active molecules such as proteins, nucleic acids or the like, after such molecules have separated within a separation matrix, using inherent properties of the molecules for their detection.
Separation of organic molecules is now routinely performed using many types of separation matrices and techniques such as in agarose or polyacrylamide gel electrophoresis. (Current Protocols in Molecular Biology (John Wiley & Sons, 1994)) A gel provides a suitably insoluble sieve so as to permit the separation of organic molecules in solution by size and conformation as they are drawn through the sieve under electromotive force. Such separation of organic molecules provides valuable insights into their structures and functions. For example, polyacrylamide gel electrophoresis (PAGE) separation can separate two polypeptides of the same size but of different isoforms or polypeptides only 100 daltons difference in size (Current Protocols, 1994, Chp. 10). Another use for PAGE is in separation of nucleic acids based on size of fragments, such as in the extremely important application of DNA sequence determination (Maniatis, "Molecular Cloning", A Laboratory Manual, 2nd ed., 1987).
When the electrophoresed molecules are labeled with a detectable signal, it is possible to detect the separations of molecules in real time. For example, nucleic acids conjugated with a fluorophore may be loaded at a first end of a polyacrylamide gel, separated under electrophoresis, and detected at a second end of the gel by laser induced fluorescence of the conjugated label. The fluorescence signal may be displayed as it is recorded or stored in a computer.
Since the first description of a real-time nucleic acid separation method and apparatus (Smith et al., Sequence Detection in Automated DNA Sequence Analysis, Nature 321: 674-679 (1986)) the technology for so-called "automated DNA sequencing" has expanded rapidly. Several automated DNA sequencing apparatuses are commercially available. Methods and apparatus for sequencing of DNA are described in U.S. Pat. Nos. 4,811,218; 4,881,812; 5,062,942; 5,091,652; 5,108,179; 5,122,345; 5,162,654; 5,171,534; 5,190,632; 5,207,880; 5,213,673; 5,230,781; 5,242,567; 5,290,419; 5,294,323; 5,307,148; 5,314,602; 5,324,401; and 5,360,523 which are incorporated herein by reference.
The means of detection of separated molecules is of fundamental technical significance in each electrophoresis system. As a general rule, most published methods of detection of electrophoresed DNA or protein samples rely on indirect detection of sample. For example, some methods rely on detection of fluorophore conjugated to the molecule of interest; other methods rely on detection of a radioactive label conjugated to the molecule of interest; still others rely on visualization of separated bands of molecules by staining of whole gels. All of these methods entail time-consuming, dangerous or cumbersome preparatory procedures for labeling the molecule of interest. For example, conjugation of a DNA sequencing primer to a fluorescein or rhodamine molecule requires use of a low efficiency conjugation reaction that leaves a large pool of unlabeled primers which reduce the efficiency of the DNA sequencing reactions, and interfere with the running of the gel.
To avoid the labeling problem, a method and apparatus are required to identify molecules based on inherent qualities of those molecules. One such method is disclosed in U.S. Pat. No. 4,930,893. This method makes use of a shearing interferometer to detect differences in the index of refraction between molecules to be analyzed and the surrounding medium. This method, however, utilizes complicated optical components, and is not shown to be useful for obtaining the real-time electrophoresis data which is important to the full implementation of genetic diagnostic and screening procedures.
It is an object of the present invention to provide an improved optical detection method for real time detection of optically active materials such as DNA or protein samples in an separation matrix based on detection of a property inherent to the DNA or protein sample.
It is a further object of the present invention to provide an apparatus for real time detection of optically active materials in a separation matrix which utilizes common optical components.